[Histonet] Extra tissues in LCM capture

From:

Yang Wang

Hi, dear histonet friends:
We need help! We have been working on the conditions for LCM for about 7
months, which is still very frustrating:-(.We tried to capture single cells
from intestinal tissues. However, the major problem we are facing now is that
we always get extra tissues from the capture. (We could pull off a whole villi
when we capture a single cellL) .We used the standard protocols provided
by “ARCTURUS” for the tissue section and dehydration.
Here is what we have done:
1.We cut 5um frozen sections and do either “pre” or “post” fixation in 4%
paraformaldehyde.
a. “Pre”: fix 2h on ice before frozen, since we are working on GFP expressing
tissue, which required pre-fixation.
b. “ Post”: fix 15-30 min after frozen
2.The slides will then be dehydrated by: 75% ethyl 30s, 95% ethy 30s, 100%
ethyl 1min and xylene 5min, then air dry 30min.
We always pull off extra tissues, which adjacent to the cell interested. We
tried to treat slides and cap with “ARCTURUS prep strip”. But it didn’t help.
We are wondering whether we need specific treatment with slides (we use fisher
superfrost plus)? Or our procedures are not proper?
Thanks a lot for help!
Yang Wang
New England Medical Center
Yang.wang@tufts.edu
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